How to process sputum samples and extract bacterial DNA for microbiota analysis

Leonardo Terranova, Martina Oriano, Antonio Teri, Luca Ruggiero, Camilla Tafuro, Paola Marchisio, Andrea Gramegna, Martina Contarini, Elisa Franceschi, Samantha Sottotetti, Lisa Cariani, Annamaria Bevivino, James D. Chalmers, Stefano Aliberti, Francesco Blasi

Research output: Contribution to journalArticle

Abstract

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

Original languageEnglish
Article number3256
JournalInternational Journal of Molecular Sciences
Volume19
Issue number10
DOIs
Publication statusPublished - Oct 20 2018

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Keywords

  • DNA extraction
  • Microbiota
  • Sequencing
  • Sputum

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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